: The applicant's long-term interests are in determining the roles of specific poxvirus genes in pathogenesis. They focus here on poxvirus-encoded proteinase inhibitors (serpins). Orthopoxviruses, including cowpox virus (CPV), encode three serpins: SPI-1, SPI-2/crmA and SPI-3. The leporipoxvirus myxoma virus (MYX) encodes two functional serpins: SERP1 and SERP2. Serpin specificity is determined largely by the identity of the P1 residue within the Reactive Site Loop (RSL) which interacts with the target proteinase. The first specific aim is to determine whether serpin pairs are functionally interchangeable in infection of rabbits and of the chicken chorioallantoic membrane. CPV crmA and MYX SERP2 both have Asp at the P1 position, and inhibit similar proteinases (caspases and granzyme B) in vitro. Preliminary studies suggest that virulence and control of primary lesion development may be separable functions of SERP2. Site-directed mutants affecting the P1 residue of crmA and SERP2 will be used to determine if any of the functions of these serpins are independent of proteinase inhibition. Experiments where SERP2 or crmA is replaced by the non-serpin baculovirus p35, an inhibitor of caspases, will evaluate the role of granzyme B in pathogenesis and lesion development. MYX SERP1 and CPV SPI-3 each have a P1 Arg residue, and very similar proteinase inhibitory properties in vitro. Replacement of SERP1 in MYX with SPI-3 changes SPI-3, normally a CPV intracellular protein, into a secreted protein. We will compare the kinetic and biological properties of SPI-3 and SERP1 proteins to investigate why the genes are not functionally interchangeable, and why SPI-3 in MYX is secreted. Specific aim 2 concerns SPI-1, which is required in rabbitpox virus for full host range. They will characterize a unique intracellular modification of SPI-1 that is dependent on a functional P1 residue, and which may represent the vestige of a serpin-proteinase complex. They have devised a genetic screen for intragenic and extragenic suppressors of a SPI-1 P14 mutation that will give information on SPI-1 structure and function. Specific aim 3 proposes coimmunoprecipitation and cross-linking studies as a means of identifying target proteinases for poxvirus serpins. They also propose genetic screens for interacting proteins based on the yeast 2-hybrid system, and on a bacterial system involving the phage and lambda repressor. Their studies are intended to provide an understanding of how poxvirus serpins function by detailing their interactions with proteinases and with other cellular and viral proteins.